Laser induced collagen crosslinking in tissue

ABSTRACT

The presently disclosed subject matter provides techniques for inducing collagen cross-linking in human tissue, such as cartilage, by inducing ionization of the water contained in the tissue to produce free radicals that induce chemical cross-linking in the human tissue. In an embodiment, a femtosecond laser operates at sufficiently low laser pulse energy to avoid optical breakdown of the tissue being treated. In an embodiment, the femtosecond laser operates in the infrared frequency range.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/893,264, filed on Feb. 9, 2018, which is a U.S. national stage filingunder 35 U.S.C. § 371 of International Application No. PCT/US2016/058353filed Oct. 23, 2016, which claims priority to U.S. provisionalapplication 62/245,805 filed on Oct. 23, 2015; U.S. provisionalapplication 62/358,035 filed on Jul. 3, 2016; and U.S. provisionalapplication 62/380,713 filed on Aug. 29, 2016. The entire contents ofeach of the above applications is hereby expressly incorporated byreference.

BACKGROUND

Collagen is an abundant protein in animals. The mechanical propertiesand structural stability of collagen based tissues, such as cartilage,tendons, ligaments, or corneal stroma, can be influenced by increasingcollagen cross-links (CXL), in the form of intra or inter moleculechemical bonds. CXL are naturally formed in tissues, but inducing new oradditional CXL can be beneficial. Strength and the ability to bend undertension are two characteristics of collagen, which is the significantcomponent in ligaments and tendons. Ligaments bind bones to bones whiletendons bind muscles to bones. The strength and flexibility of collagenprovides for ease of movement. Strength and flexibility are also twocharacteristics of cartilage, which covers the ends of bones at a joint.Cartilage allows one bone to glide over another as it protects andprevents bones from rubbing against each other.

BRIEF SUMMARY

The presently disclosed subject matter provides techniques for inducingcollagen cross-linking in human tissue, such as cartilage or cornea, orskin, without using a photosensitizer (e.g., riboflavin). While laserlight at any frequency can be used, in some embodiments a laser outsideof the ultraviolet (UV) frequency band is used. In an embodiment, afemtosecond laser operates at sufficiently low laser pulse energy toavoid optical breakdown of tissue. In an embodiment, the femtosecondlaser operates in the infrared frequency range.

Advances in femtosecond lasers enable using femtosecond laser emittingvisible wavelength to ionize liquid water. Femtosecond pulsed lasersalso enable direct observation of products of water ionization anddissociation and other aqueous media.

It has been experimentally shown that a scenario, low-density plasma isformed, and treatment is reduced to ionization and dissociation of thewater content within the focal volume. This treatment also results inproduction of reactive oxygen species. Advantageously, the producing ofreactive oxygen causes has a disinfecting effect. Initially, ionizationof the water molecule occurs, and the ejected electron is quicklyhydrated resulting in formation of a solvated electron. Further, thecation radical of water, H₂O+, is rather unstable and reacts with awater molecule producing hydrogen ion H₃O⁺, and hydroxyl radical OH*.Concurrently dissociation of the excited water molecule occursH₂O*^(→)H+OH*, though other reactive oxygen species may also beproduced.

This disclosure expands use of low-density-plasma from sub-cellularlevel to a microscale. Scaling up has potential to enable microscopicalteration of the chemical composition of target material. In someembodiments, the target material is connective tissue in the human body.In an exemplary embodiment, the tissue is corneal tissue, which in turnresults in overall changes of its properties. In other embodiments, thetarget material is a cartilage. In other embodiments the tissue istendon, ligament, or corneal stroma. A femtosecond oscillator coupledwith an optical delivery system can be adjusted in such a way to deliverlocal, spatially resolved alteration of chemical composition of tissuewithout any harmful influences of thermal stresses, such as collagendenaturation. In the case of ocular media, corneal hazing can beavoided.

In embodiments of the disclosed subject matter, the tissue isstrengthened selectively to correct defects. In the case of cornea,defects such as keratoconus can be corrected by strengthening thecornea. In further embodiments, the corneal curvature is modified tocorrect vision problems such as myopia. In still further embodiments,cartilage is treated with a laser to induce cross-linking and therebystrengthen the cartilage. The strengthening of cartilage can slow theprogression of, or reverse osteoarthritis.

In embodiments, a laser is scanned over a selected three-dimensionalregion, which may be a continuous region or a discontinuous region ofthe cornea to be modified to generate a selected shape change. In amethod, the cornea shape is measured and then a pattern of illuminationsselected to change the shape of the cornea toward a target is generatedin a controller database. In embodiments, the laser can be a femtosecondlaser. In embodiments, the femtosecond laser can be a Nd:Glassfemtosecond laser. In embodiments, the femtosecond laser can output anaverage power from about 10 mW to about 100 mW. In embodiments, thefemtosecond laser can have a pulse energy of from about 0.1 nJ to about10 nJ. In embodiments, the cornea can receive from about 10 mW to about100 mW infrared irradiation from the light source.

In embodiments, the laser can be scanned in a pattern of exposurecomprising a circle, annulus, and/or ellipse. The laser can be scannedin multiple layers of the cornea.

According to another aspect of the disclosed subject matter, systems ofreshaping curvature of are cornea are provided. In embodiments, anexample system of reshaping curvature of a region of a cornea having aninitial curvature can include illumination optics configured to projectan illumination pattern onto at least a portion of the cornea and acamera configured to record a pattern reflection from the at least aportion of the cornea. The system can also include a control system,coupled to the camera, configured to convert the pattern reflection to acorneal topography, and configured to compare the corneal topography toa desired corneal topography to determine a deformation map 302. Thesystem can further include a laser system, configured to induceionization in the region of the cornea according to the deformation map301 to reshape the region from the initial curvature to a new curvatureand a coupling device, configured to stabilize the laser system withrespect to the cornea.

In embodiments, the laser system is configured to induce cross-linkingof collagen in the cornea according to the deformation map. Inembodiments, the laser system can include a femtosecond laser. Thefemtosecond laser can have a pulse width of from about 50 to 150femtoseconds (fs). The femtosecond laser can have an average power fromabout 10 mW to about 100 mW. The femtosecond laser can irradiate lightin the wavelength range from about 600 nm to about 1100 nm.

In embodiments, the laser system includes a high magnification objectivelens and a galvanometer configured to raster a laser beam. The lasersystem can further include an attenuator.

According to another aspect of the disclosed subject matter, anapparatus for adapting a laser system for reshaping curvature of aregion of a cornea having an initial curvature is provided. Inembodiments, an example apparatus can include a control system, adaptedto be coupled to the laser system and configured to compare an existingcorneal topography of at least a portion of the cornea to a desiredcorneal topography to determine a deformation map. The system canfurther include laser modification optics, coupled to the control systemand configured to adjust laser output of the laser system, to modify aregion of the cornea according to the deformation map.

Additional features and advantages of the application will be describedhereinafter which form the subject of the claims.

In embodiments, the laser system is adapted to induce cross-linking inconnective tissue, such as cartilage.

In embodiments, the laser system and the regime of controlling theoutput of the system irradiates connective tissue such that thewavelength of the laser is not absorbed by the targeted tissue itself,but rather by water in and around the tissue. Thus, optical breakdown ofthe tissue is avoided, but the laser is controlled to cause ionizationof the water molecules, which in turn generates free radicals which theninduce cross-linking in the tissue. This approach is different fromdirectly inducing cross-linking in tissue, because tissues generallyabsorb only a limited set of wavelengths, which would limit theimplementation of treatment system. Conversely, water can be ionized bya broad spectrum of wavelengths to generate free radicals and to avoidbreakdown of the targeted tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a diagram of an exemplary system in accordance withthe disclosed subject matter.

FIG. 2 illustrates a diagram of exemplary topography controls.

FIG. 3 illustrates a diagram of exemplary cross-linking controls.

FIG. 4 illustrates a diagram of an exemplary cross-linking system.

FIG. 5A illustrates a flow diagram of an exemplary cross-linking processapplied to the cornea.

FIG. 5B illustrates a flow diagram of another exemplary cross-linkingprocess applied to cartilage.

FIG. 6 illustrates a possible cross-linking mechanism for reshapingcornea using a laser.

FIG. 7A illustrates a diagram an exemplary system for providing multiplebeam exposure.

FIG. 7B illustrates a diagram another exemplary system for providingmultiple beam exposure.

FIG. 7C illustrates a diagram of an exemplary scanning objectivessystem.

FIG. 8 illustrates an exemplary setup for Example 1 described below.

FIG. 9 demonstrates a schematic diagram of loading regime during theinflation test.

FIG. 10A demonstrates an initial shape of the cornea assessed by DIC.

FIG. 10B demonstrates an oblique reconstruction of the cornea by DIC.

FIG. 11 demonstrates displacement maps of the control cornea in zdirection at different pressures.

FIG. 12 demonstrates displacement maps of the half-treated cornea in zdirection at different pressures.

FIG. 13 demonstrates loading-displacement hysteresis curves for thecontrol cornea.

FIG. 14 demonstrates loading-displacement hysteresis curves for the halftreated cornea and the control cornea at 0.019 kPa/s pressurizationrate.

FIG. 15 demonstrates loading-displacement hysteresis curves for the halftreated cornea and the control cornea at 0.15 kPa/s pressurization rate.

FIG. 16 shows Raman spectra of a treated and a control region of thehalf-treated cornea.

FIGS. 17A-17C illustrate the results of the disclosed method and systemapplied to porcine eye shape.

FIGS. 18A-18B illustrate a plot of average lasing energy and numericalaperture against irradiance level of an embodiment.

FIGS. 19A-19B illustrate results obtained via electro paramagneticresonance spectroscopy of an embodiment.

FIG. 20 illustrates a temperature distribution as a function of distancefrom the focal volume of an embodiment.

FIG. 21 illustrates a stereographic micrograph of a porcine cornea thathas been treated according to an embodiment.

FIGS. 22A-22B show results of treatment of cartilage.

DESCRIPTION

The presently disclosed subject matter provides methods and systems forinducing collagen cross-linking in tissues. In embodiments, the focusedlaser light provides strengthening of the corneal collagen andmodification of the corneal curvature. The cross-linking is induced byinducing ionization in the tissue. The ionization can be induced by alaser and can create cross-links. Specifically, ionization of water inor around the tissue generates free radicals which in turn inducecross-linking in the tissue, without optical breakdown in the tissue.

A femtosecond oscillator coupled with an optical delivery system can beadjusted in such a way to deliver local, spatially resolved alterationof chemical composition of ocular media without any harmful influencesof thermal stresses, such as collagen denaturation and corneal hazing. Alow-density plasma is formed in response to irradiation of a target areaby the optical delivery system, and treatment is reduced to ionizationand dissociation of the water content within the focal volume. Thistreatment results in production of reactive oxygen species. Initially,ionization of the water molecule occurs, and the ejected electron isquickly hydrated resulting in formation of a solvated electron. Further,the cation radical of water, H₂O+, is rather unstable and react with awater molecule producing hydrogen ion H₃O⁺, and hydroxyl radical OH*.Concurrently dissociation of the excited water molecule occursH₂O*^(→)H+OH*. Experiments have captured the resulting OH*, but otherreactive oxygen species may also be produced.

The disclosure restricts the laser regime such that the treatment isalways below the optical breakdown, and thus relies on the ionizationpotential for alteration of corneal geometry or the generation ofcross-links in other tissue, such as cartilage. If a femtosecond laseroperates below the energy level required for optical breakdown,ionization of atoms within the focal volume is possible. The ionizationprobability has a number of resonance maxima due to intermediatetransition of the atom to an excited state. In the vicinity of suchmaximum the ionization cross-section increases by several orders ofmagnitude enabling ionization even if the frequency of the incomingelectromagnetic wave is lower than the ionization potential. Tests(including spin trapping characterization) confirm that such a scenarioenables creation of singlet oxygens, which likely react with the freecarbonyl groups in the collagen chains. Such reactions result incrosslink formation. In a particular test, dityrosine crosslink wasformed after femtosecond oscillator treatment of L-tyrosine solution.

The methods and systems can be used to treat various corneal disordersincluding keratoconus, myopia, hyperopia, stigmatism, irregularastigmatism, and other ectatic diseases (e.g., those that result from aweakened corneal stroma). The methods and systems can also be used inrefractive surgery, e.g., to modify corneal curvature or correctirregular surfaces and higher order optical aberrations. The methods andsystems can also be used to induce cross-linking in cartilage fortreatment of osteoarthritis or other connective tissue disorders.

As embodied herein, ionization can be created within tissue using alaser emission that is absorbed by the tissue. For example, the laseremission can be based on ultrashort laser pulses. As used herein, thephrase “ultrashort laser pulses” includes emissions in the femtosecond,picosecond, and nanosecond ranges. Nonlinear absorption of laseremissions can occur in part due to the highly compressed nature of thelight pulses, allowing treatments of the interior of a transparentdielectric, such as corneal tissue, without affecting the surface layer.

The ultrashort laser pulse can induce low-density plasma that ionizeswater molecules within the tissue, but the laser operates below theenergy level required for optical breakdown. Optical breakdown is theeffect of an ultrafast laser focused in the interior of collagen richtissue, where photoionization triggers non-linear absorption. Continuedsupply of incoming photons leads to the buildup of free electrons,further leading to avalanche ionization, which enhances the growth offree electron density resulting in formation of plasma. As contrastedfrom the low-density plasma, high-density, opaque plasma stronglyabsorbs laser energy through free carrier absorption. The high-densityplasma expands rapidly, creating a shock-wave which propagates intosurrounding material, creating optical breakdown.

Collagen cross-linking can be safely induced when the laser is operatedbelow optical breakdown level in the so-called “low-density plasma”regime. For example, the laser emission, as defined by its wavelength,temporal pulse width, and pulse energy, as well as the numericalaperture of the scanning objective and the scanning speed should be highenough to induce ionization of water molecules in the collagen richtissue, but below optical breakdown level. Further, such ionization canbe induced in the cornea without reducing the transparency of thecornea.

Various parameters of the laser can be manipulated to control the safetyand efficiency of the cross-linking of the collagen. For example, thelaser beam, as defined by its wavelength, temporal pulse width, andpulse energy, as well as the numerical aperture of the scanningobjective and the scanning speed should be high enough to induceionization of water molecules in the cornea, but below optical breakdownlevel. Accordingly, these parameters can be maintained within certainranges.

Without being bound to a particular theory, the ionization can cause theformation of reactive oxygen products, such as singlet oxygen, OH⁻, andH₂O₂, which in turn can interact with collagen and increasecross-linking in the fibrils, as shown in FIG. 6 . Additionally, singletoxygen generated by the ionization can inactivate collagenase and have agermicidal effect, increasing the utility of these methods for clinicalapplications. In embodiments, deuterium oxide can be introduced onto thecornea to prolong half-life of the produced singlet oxygen, therebyincreasing cross-linking efficiency.

In certain aspects, the presently disclosed subject matter providesmethods of inducing such ionization. The methods can be used in thetreatment of various ectatic diseases or during refractive surgery. Themethods can include modifying the corneal curvature by inducingselective corneal cross-linking.

Corneal deformations can be induced using a laser emission, as describedin further detail below. Corneal deformations can be selectively inducedusing a patterned laser exposure. The pattern of exposure can depend onthe desired deformations, and can be customized to the patient. Inembodiments, the method can include mapping the topography of thepatient's eye prior to treatment and designing a pattern of exposurebased on that topography.

For the purpose of illustration and not limitation, FIG. 1 is aschematic representation of an exemplary system according to anon-limiting embodiment of the disclosed subject matter having twosubsystems: a Topography System 100 and a Cross-linking System 200. Forthe purpose of illustration and not limitation, FIG. 2 is a furtherschematic representation of the Topography System 100.

As shown in FIG. 1 , an embodiment of the cross-linking system 200includes an objective 201. The objective 201 can be a scanning objectivewith a large numerical aperture. The large numerical aperture allows theobjective 201 to focus diffuse light to a small area. A laser 203supplies the light (laser light) to the objective 201. In an embodiment,one or more optical filters 202 may be interspersed between the laser203 and the objective 201. The laser 203 can be a femtosecond laser thatoutputs laser light. In some embodiments, the laser light has a singlefrequency, and in other embodiments includes multiple frequencies.Embodiments may use any wavelength including multiple or continuousspectra covering a wide range of wavelengths. However in embodiments,preferably radiation at frequencies that may harm tissue or reduce thelocality of the generation of reactive species are minimized oreliminated. Radiation that may be directly absorbed by the collagen maybe minimized or eliminated. In an embodiment the frequency orfrequencies of the laser 203 are outside of the ultraviolet range. Inembodiments, the frequency or frequencies of the laser 203 are in theinfra-red frequency band. The laser 203 receives control input fromcontrols 400, which may be implemented on a stand-alone processingdevice or as embedded circuitry of the system.

As further shown in FIG. 1 , the objective 201 focuses incoming laserlight into a focused beam 601 which irradiates a target. In the exampleof FIG. 1 , the target is tissue 411. In embodiments, the tissue 411could be a cornea 402, cartilage 405, or other tissue. FIG. 4 .illustrates embodiments with cornea 402 and cartilage 405. The objective201 may have a large numerical aperture. In an embodiment, the numericalaperture is 0.6, with a long working distance.

Referring still to FIG. 1 , a topography system 100 includes controls300 which communicate with controls 400 of the cross-linking system 200.The topography system 100 includes a light source 104 and an imagingdevice, such as a camera 103. The light source 104 projects light tomirror 102 and a device, such as a mask, to produce an illuminationpattern 101. The illumination pattern 101 guides the cross-linkingsystem 200 to induce cross-linking in specified locations to produce thedesired change in the treated tissue.

Referring to FIG. 2 , additional details of the controls 300 of thetopography system 100 are shown. A spatial deformation map 302 definesspatially the deformation of the cornea, which, when considered with thetopography map 301 of the cornea, provides information on where toinduce cross-linking.

Referring to FIG. 3 , a skilled artisan will understand how to create atopographical map 301 of the cornea. By comparing the topographical mapof the patient's cornea to a desired corneal topography, the controlsystem 300 then generates a spatial differential map 305. As shown inFIG. 3 , a mathematical elemental model 401 may be used to compareexisting corneal topography to desired corneal topography to define adeformation map 403.

In embodiments, the illuminated pattern 101 can be a series ofconcentric rings. In embodiments, the control system 300 can be aprocesser coupled to a memory and further coupled to a storage device.

As embodied herein, a laser can induce ionization to cause cornealcross-linking. For the purpose of illustration and not limitation, FIG.4 is a further schematic representation of a non-limiting embodiment ofa system in accordance with the disclosed subject matter. Inembodiments, the control system 400 is linked to a laser 203 and furtherlinked to one or more scanning objectives 201. In case of multiplescanning objectives 201, the set of scanning objectives 201 can enablethe laser 203 to raster a pulsed beam across predetermined regions ofthe patient's cornea through the coupling device. The regions of thepatient's cornea to be scanned in raster fashion can be related to thedeformation map 302 generated by the control system 400, or anotherpattern of exposure designed for the treatment. The cross-linking causedby the ionization induced by the laser can create corneal stiffeningthat alters the dimensions of corneal curvature in a predictable way.

Referring again to FIG. 4 , the scanning objective 201 can be alsofocused on cartilage 405 to induce cross-linking in such tissue. In thisexample, the deformation map 403 of a cornea would not be used, butinstead a different pattern would be used to illuminate the cartilage405. While not illustrated in FIG. 4 , in certain embodiments thescanning objective 201 may be replaced or supplemented with an opticalpathway to guide the laser light to the treatment tissue. In anembodiment, the optical pathway may be an endoscope that is used toguide the laser light to cartilage in a live patient

In embodiments, the cornea can be scanned in raster fashion with thelaser beam in multiple layers. For example, the laser beam can scan thecornea 402 in a first pattern of exposure, and then scan the cornea in asecond pattern of exposure. The first pattern of exposure and the secondpattern of exposure can wholly or partially overlap on the cornea toprovide multiple layers of exposure. Using a high-aperture canningobjective 201 enables the application of energy at different specifieddepths in the cornea 402.

Referring to FIG. 5A, an example of a method of inducing cross-linkingin corneal tissue is shown. In step 501, the topography of the patient'scornea is measured. In step 502, the desired cornea geometry iscomputed. In some embodiments, the goal may to strengthen the corneawithout changing its shape. In this case, step 502 computes the desiredlocations to strengthen without changing the corneal shape. In anembodiment, a coupling mechanism may be placed over the eye to betreated in step 503. However, this method is not limited to suchembodiments, and the cornea may be treated without a coupling mechanismalso. In step 504, the laser 203 is driven to emit low energy pulseswhich are guided and focused by the cross-linking system 200 discussedabove. As shown in step 505, the interaction of the pulse laser with thetreated tissue and/or the aqueous medium in and around the tissueinitiates cross-linking. In step 506, a lens of the coupling mechanism(if one was used) is removed from the cornea.

In embodiments, the techniques of the present disclosure can be used tomodify corneal curvature. For example, these techniques can be used incorneal flattening, i.e., to reduce the optical power of the cornealsurface. In embodiments, the cornea 402 can be flattened by controllingthe spacing and layering of laser pulses within a central treatmentzone. For further example, these techniques can be used in cornealsteepening. By way of example, and not limitation, the pattern caninclude one or more circles, ellipses, annuli, and combinations thereof,which can be chosen based on the desired shape change. For example, acircular treatment pattern can be employed to flatten the curvature ofthe cornea. An annular (e.g., toric) treatment pattern can be employedto steepen the curvature of the cornea. Additionally, the density of thetreatment (i.e., the points of exposure) can be modulated.

Moreover, certain patterns of exposure can be used to treat certaindisorders. For example, an elliptical shape, which can be disk-shaped ortoric, can be used to treat astigmatism. The elliptical pattern can bedesigned such that one meridian of the astigmatism is flattened morethan the other. During treatment, the elliptical pattern can be placedover the corneal projection of the entrance pupil and aligned such thatthe astigmatic axis of the pattern overlies the astigmatic pattern onthe cornea. As such, the steeper axis of the astigmatism can beflattened more than the flatter axis to correct the astigmatism.

Additionally, the pattern of exposure can be used to treat cornealinfections and melts. The ionization induced by the pattern of exposuremay be employed to kill bacteria, fungi, and infected cells. Forexample, first, the extent of the infection and/or melt can bedetermined using various methods as known in the art, including slitlamp bio microscopy, fluorescein staining, optical coherence tomography(OCT) analysis of the corneal structure, and other conventional clinicaltechniques. Then, the corneal tissue can be treated in multiple layersto cover the volume of damaged stroma and/or infectious elements.

In addition to the shapes previously described, the pattern of exposurecan further include additional shapes including bow-ties and concentricrings. Additionally, or alternatively, the pattern can include acustom-designed shape. For example, in embodiments, a custom-designshape can be designed to correct a higher order aberration. Inembodiments, a custom-designed shape can be based on the corneatopography. In particular embodiments, for example, where the patienthas an uneven corneal topography, a custom-designed shape may bepreferred.

Thus, as embodied herein, it is not necessary to provide a physicaldevice to mechanically induce the deformations prior treating the corneawith the laser emission. However, in embodiments, a physical device,such as a suction contact lens, can be used to induce cornealdeformations in conjunction with laser beam treatment. The device can becustomized such that the back of the device forms a mold for the desireddeformations, i.e., based on the deformation map 302.

FIG. 6 illustrates conceptually the change in corneal shape after thecornea is exposed to focused beam 601 of pulsed laser light. Collagen602 is naturally present in the cornea. The application of focused beam601 results in the production of free radicals from water in thetreatment area, without causing optical breakdown in the corneal tissue.The free radicals induce cross-linking, shown conceptually ascross-links 604 in the reshaped cornea 402 b. The induced cross-linkscan change the shape as well as the elasticity of the cornea (e.g., tocounteract keratoconus.)

Referring to FIG. 5B, an example of the process of inducingcross-linking in cartilage is illustrated. This process has somesimilarities with that shown in FIG. 5A. In step 507 the cartilage isassessed to determine its condition. In step 508, the desiredlocation(s) and amount of treatment is computed. The treatment can bespread out over a large area or focused on only specific areas. Thetreatment can be varied by controlling the power and pulse rate of thelaser 203. In step 509 access is provided to the cartilage to betreated. In an embodiment, this may be through surgical incisions toprovide access to an endoscope. An endoscope may guide the pulsed laserlight through an optical pathway to the target area. In step 510 lowenergy pulses from a femtosecond laser are applied to the desiredtreatment location(s), with the scanning objective 201 possibly movingor scanning over the treatment area. In step 511, crosslinking isinitiated in the cartilage tissue in response to the laser treatment.

In embodiments described previously, the laser can emit ultrashort lightpulses, e.g., with a temporal length below 1 nanosecond. Generation ofsuch short pulses can be achieved with the technique of passive modelocking. The laser 203 can be any suitable laser type, including bulklasers, fiber lasers, dye lasers, semiconductor lasers, and other typesof lasers. In an embodiment, the laser operates in the infraredfrequency range. In other embodiments, the lasers may cover a wide rangeof spectra domain. In embodiments, the disclosed subject matter can beimplemented as an add-on system to a femtosecond laser system, such asused in certain Lasik systems.

As embodied herein, the operating parameters of the laser can be varieddepending on certain environmental factors. By way of example, and notlimitation, such environmental factors can include the nature of theinterstitial fluid, the presence and amounts of dissolved nutrients, theosmolarity, the humidity, and the oxygen levels. For example, certainenvironmental factors can impact, e.g., the thickness of the cornea ortreatment efficiency. Thus, in embodiments, the operating parametersand/or pattern of exposure can be modulated accordingly.

In embodiments, and with further reference to FIGS. 1 and 4 , thescanning objective(s) 201 can include a high magnification, e.g. 40×,objective lens and a galvanometer for scanning the laser across thecornea. The objective lens 201 can have a high numerical aperture, e.g.,0.6, with a long working distance. The objective can be interchangeableto accommodate for use of a range of numerical apertures as well asworking distances.

In embodiments, the scanning objectives can include multiple mirrors onan automated track, as illustrated in FIG. 7C. For the purpose ofillustration and not limitation, FIG. 7C is an exemplary schematicrepresentation of the scanning objectives. The laser light can bedirected along the x axis. Mirror A (702) can travel along x-axis track.At a determined x position, mirror A can deflect the incoming laserlight orthogonally into they plane. Mirror B (704) can be adjusted suchthat its x position is the same as that of mirror A. Mirror B can thentraverse along they axis to a desired y position to deflect the laserlight orthogonally into the z plane towards the objective lens. Theobjective lens and mirror B can be housed on the same posting and movetogether. Traversing the track can be done continuously or discretelythrough DC servo motor control along the rails. The scanning of thecornea with the laser beam can be accomplished by moving the mirrors,according to the control system 400 The mirrors and lens are constructedwith wavelength and energy appropriate materials known in the art.

In particular embodiments, the laser can be a Nd:Glass femtosecondlaser. In embodiments, the laser wavelength can be in the range fromabout 250 nm to about 1600 nm. In embodiments, the femtosecond laser canhave a temporal pulse width of from about 20 fs to about 26 ps. Inembodiments, the pulse energy is from about 0.1 nJ to 100 nJ, 0.1 nJ toabout 50 μJ, 0.1 nJ to about 10 μJ, from about 0.5 nJ to 50 nJ, or fromabout 1 nJ to 10 nJ. In embodiments, the femtosecond laser can be aSpirit femtosecond laser in combination with a Spirit-NOPA™ amplifier(Spectra-Physics, Santa Clara, Calif.). The numerical aperture of thescanning objective can range from about 0.05 to about 1. In embodiments,the numerical aperture can be selected based on the pulse energy, e.g.,by balancing the numerical aperture and the pulse energy. The laser beamcan be stationary or moving. Where the laser beam is moving, thescanning speed can be any suitable scanning speed based on the equipmentbeing used.

As embodied herein, the laser can provide one or more beams. Thus, thepresently disclosed techniques can be used to provide a multiple beamexposure. Multiple beam exposure can increase the treatment area and/ordecrease the length of exposure. For example, treatment with multiplebeams can decrease the exposure time to less than an hour. Inembodiments, treatment with multiple beams can decrease the exposuretime to less than 5 minutes per layer, e.g., 2 to 3 minutes per layer.

In embodiments, the multiple beams can be provided by splitting a laserbeam to multiple scanning objectives. For example, a laser head caninclude multiple scanning objectives bundled together, as shown in FIGS.7A and 7B. FIG. 7A illustrates an example of a linear array 710 ofobjectives 701. FIG. 7B illustrates a two dimensional array 711 ofobjectives 701. Although the objectives 701 are illustrated as identicalin the drawings, in embodiments different objectives are used atdifferent positions in the array. A high energy laser beam (e.g., havinga pulse energy of greater than about 10 μJ) can be split using a beamsplitter to send individual laser beams to each scanning objective.Therefore, the number of passes required to fully treat the cornea canbe reduced by providing multiple laser beams simultaneously. Inembodiments, an entire corneal layer could be treated simultaneously,e.g., by bundling many scanning objectives to the laser head such thatonly one pass is required.

The following examples are merely illustrative of the presentlydisclosed subject matter and should not be considered as limiting in anyway.

Example 1: Use of Femtosecond Laser to Cross-Link Porcine Cornea

This Example illustrates the effect of femtosecond laser irradiation toa porcine cornea.

Porcine eyes were obtained from a commercial supplier (AnimalTechnologies, Tyler, Tex.). The eyes were harvested and frozen within 3hours after slaughter. The eyes were carefully thawed immediately beforethe example was performed. The globe was mounted onto the metal ring andfixed with cyanoacrylate, such that the cornea was placed at the centerand left exposed after the mounting excess tissue was removed.Subsequently, the upper surface of cornea was moistened with phosphatebuffered saline (PBS) solution and a coverslip was placed on top of thespecimen to reduce light scattering from the laser. Placement of thecoverslip also ensured the flatness of the top surface of the cornea.The metal ring with cornea was fixed onto a 3-axis motorized translationstage using a custom made holder.

A Nd:Glass femtosecond laser system was used to generate laser pulseswith temporal pulse width of 99 fs and 52.06 MHz repetition rate at1059.2 nm wavelength. A Zeiss Plan Neofluar 40×/0.6 objective lens wasemployed to focus the beam, and the pulse energy was measured to be 60mW after the objective lens. The laser beam was focused in the interiorof cornea, creating planar zigzag patterns with 50 μm pitch at feed rateof 1 mm/s. Multiple planes parallel to the corneal surface were treatedwith 150 μm distance between two consecutive planes. During this processcornea was moistened with PBS solution to prevent drying. A schematic ofthe example setup is shown in FIG. 8 .

Changes in the chemical composition of the corneas were assessed usingRaman spectroscopy. Raman spectra were acquired with a confocalmicro-spectrometer (Renishaw InVia), equipped with 1800 gr/mm. Incidentlaser excitation was provided by helium-neon laser with 632.8 nmwavelength, delivered by a 100× objective. Raman signal was acquired by30 accumulations, each lasting 10 seconds.

An inflation test was used to provide information about the changes inthe mechanical properties of the corneas subjected to femtosecond laserpulses. A loading regimen consisting of a series of linear load/unloadcycles was utilized on eight specimens to characterize the change in themechanical properties of cornea subjected to femtosecond lasertreatment. Each sample was initially subjected to two pressure linearload-unload cycles to determine the effects of pre-conditioning. Thesecycles were parted by a 15-minute recovery period. (FIG. 9 ).Pressure-displacement curves obtained from pre-conditioning werecompared against one another and against the identical post-conditioningcycle to verify that the sample did not degrade during the test.Baseline pressure used was 0.5 kPa, the lowest pressure able to supportthe specimen without buckling. The maximum pressure used was 5.4 kPa.Pre-conditioning cycles were followed by three loading cycles, in whichloading rate was varied. The first test had loading rate identical tothe preconditioning, 0.15 kPa/s, and the latter two tests had loadingrates of 0.019 kPa/s and 0.15 kPa/s, respectively.

Stereoscopic digital image correlation (DIC) system was employed toacquire spatially and time resolved displacement maps of the cornealsurface during the inflation tests. Two cameras were located above thesample, at 15° angle with respect to the vertical axis. The image pairswere analyzed with 3D DIC software package (VIC 3D Correlated Solutions,Inc.). The reference configuration as a function of Z-heights for amatrix of x and y coordinates (FIG. 10 ), as well as deformation fieldsfor each subsequent image-pair were extracted by the DIC algorithm. Thealgorithm was capable of providing displacement components U-, V-, andW-corresponding to the deformation along the x-, y-, and z-axes of thecamera coordinate system. These axes were aligned with the optical axesof the cornea. For this example, only the deformation in the directioncoincident with corneal bulging (W-displacement) was of interest. Thenasal-temporal, the inferior-superior and the thickness at the center ofeach specimen were measured with caliper before and after the inflationtest.

FIG. 11 shows examples deformation maps (such as a spatial deformationmap 302) of porcine cornea at various pressures during the inflationtest. Each image frame corresponds to one time step during themulti-cycle loading regimen. The deformation shown is along the z-axis,which coincides with the optic axis of the cornea. Specifically, theimage frames shown correspond to the first loading regime and similartrends were observed when different loading rates were applied. Only oneset of data is shown.

While FIG. 11 shows data from the control cornea, FIG. 12 depicts theresults of the corneal tissue, half of which was treated with thefemtosecond laser. Nonzero values on the edges of DIC maps occur due tothe condition of the DIC method to have a finite analysis window.Results shown correspond to the inflation regime characterized with 0.15kPa/s rate. FIG. 11 also shows the deformation of the control cornea isaxisymmetric. The apex displacement, defined as maximum out-of-planedisplacement in the central part of the cornea, reaches value of 0.361mm at 5.5 kPa pressure. The viscoelastic response of the cornea thatresembles J-shaped pressure-displacement curve is observed. (FIG. 13 ).When the half-treated cornea is subjected to identical loadingconditions, treated part of the corneal tissue exhibits less deformationthan the untreated portion. The displacement in the entire corneaincreases with the rise of the inflation pressure, but the deformationof the treated part of the corneal tissue is much lower than theuntreated region. Maximum deformation in the untreated region is lowerthan the apex deformation in the control cornea.

The difference in the observed displacement between untreated andtreated regions of the corneal tissue can be attributed to stiffening ofthe laser treated part of the cornea, which can be attributed tocreation of cross-links that increase the structural stability of thestroma. The stiffened (i.e., treated) region required more pressure fordisplacement. The pressure-displacement curve was constructed byextracting the apex displacement at each time step of the inflation test(FIG. 13 ). For the control specimens, apex displacement was used,whereas in the case of half-treated sample, representative points inboth treated and untreated region were utilized. Two differentpressurization rates are compared.

FIGS. 14 and 15 show hysteresis curves obtained from untreated andtreated part of the porcine cornea, subject to 0.019 kPa/s and 0.15kPa/s pressurization rates, respectively. The change in the relativeslope of the hysteresis curve indicates stiffening of the cornealtissue, as more pressure was required to displace the treated tissue.The nonlinear ionization induced within the focal volume by femtosecondoscillator can create singlet oxygen, which then reacts with the freecarbonyl groups subsequently forming the CXLs, which can be responsiblefor the observed corneal stiffening.

Raman spectra of the untreated and treated part of the cornea (FIG. 16 )show difference in the chemical composition after laser irradiation. TheRaman signal was normalized with respect to phenylalanine peak locatedat 1000 cm⁻ The Raman band associated with carbonyl group (1670 cm⁻¹)and the peak associated with Amide III (1265 cm⁻¹) are diminished in thetreated part of the cornea, which is consistent with cross-linkingbecause the ionization results in the formation of singlet oxygen thatcan then react with free carbonyl groups to form the cornealcross-links.

FIGS. 17A-C illustrate the results of the disclosed method and systemapplied to porcine eye shaped. FIG. 17A is an elevation map depictingtopography of the cornea of a porcine eye before treatment, and FIG. 17Bis an elevation map depicting topography of the cornea after treatment.The difference between the two can be more easily quantified byreferring to FIG. 17C, which illustrates the difference in the effectiverefractive power of the two.

Electron Paramagnetic Resonance (EPR) Spectroscopy

Spin-trapping reagent 5,5-dimethy-1-pyrroline-N-oxide (DMPO, CaymanChemicals, USA) was solved in Dulbecco's phosphate-buffered saline(DPBS), with final concentration of 100 mM, just before the treatment.170 μL of the solution was placed into a shallow dish (2×8 mm) and putonto the 3-axis motorized stage for treatment. A control sample wasconcurrently prepared by following the same procedure. A Nd:Glassfemtosecond laser system was used to generate laser pulses with temporalpulse width of 99 fs and 52.06 MHz repetition rate at 1059.2 nmwavelength. A Zeiss Plan Neofluar 40×/0.6 objective lens was employed tofocus the beam, and the pulse energy was measured to be 60 mW after theobjective lens. Immediately after the treatment the solution wascollected into 0.5 mL tubes, which were then placed into liquid nitrogencanister and transported to EPR spectrometer (Bruker BioSpin GhbH EMXElectron Paramagnetic Resonance Spectrometer, Bruker BioSpin GmbH,Germany). Sample transport time between the treatment and EPR analysisnever exceeded 15 minutes. The control samples were also collected into0.5-ml tubes, transported in liquid nitrogen and characterized forcomparison.

Temperature and Refractive Index Measurements

Porcine eyes were obtained from a commercial supplier (e.g., AnimalTechnologies, Tyler, Tex.). The eyes were harvested and flash frozenwithin 3 hours after slaughter. The eyes were carefully thawedimmediately before the experiments. Corneas were harvested and mountedonto a custom built holder. After the mounting, excess tissue wasremoved and cornea placed onto the 3-axis motorized stage. Subsequently,cornea tissue was moistened with phosphate buffered saline (PBS)solution. The cornea was then punctured with needle-like head of acustomized thermocouple (temperature measurement range 0-100° C.). Thetip of the thermocouple was inserted in the middle of the harvestedcorneal tissue. Real-time temperature readings were displayed on a LEDmonitor. The above-described Nd:Glass femtosecond laser system wasemployed for the treatment. The focal point was aligned with the tip ofthe thermocouple and temperature distribution measured as the focalvolume was circulated around the tip of the thermocouple. In addition totemperature measurements, potential refractive index changes in cornealrefractive index were examined. In the central part of the cornea 3×5 mmrectangular area was treated following the same protocol. After thetreatment, mounted cornea was examined with stereographic microscope aswell as transmission microscope (Olympus, Japan) equipped with NomarskiInterference Contrast (NIC) prism to enhance contrast between regions ofthe cornea that may have different refractive index. Imaging was laterrepeated on fresh corneas.

Measurements of Photoionization

Treatment of transparent biological media by femtosecond lasers can beachieved at least due to nonlinear nature of laser-matter interaction,which results in formation of quasi-free electrons via photoionization.Photoionization in transparent dielectrics can be achieved either viamultiphoton ionization (MPI) or tunneling ionization. In the former, anelectron absorbs several photons simultaneously, whereas the latter ischaracterized by the electromagnetic field that is strong enough todistort the Coulomb well so that the electron can escape the energybarrier.

Free electrons produced by MPI or tunneling ionization gain kineticenergy by absorbing photons in a process called ‘inverseBremsstrahlung’. The process includes collisions with heavy chargedparticles (ions or nuclei), which are needed for energy and momentumconservation. A sequence of inverse Bremsstrahlung events results inincrease of the electron's kinetic energy that is now sufficient toproduce another free electron via impact ionization. The sequence isrepeated, resulting in growth of the free electron density in a processthat resembles an avalanche.

Femtosecond laser-assisted treatment of ocular media can includephotodisruption, which relies on formation of cavitation bubble(s)within the focal volume to create incision(s) in the interior of acornea. Achieving an optical breakdown in the laser focus inducescavitation bubble.

It has been experimentally shown that this threshold in ocular andbiological media is similar to the optical breakdown threshold in water.Since the number of free electrons produced during a single pulse is afunction of irradiance, one could couple a femtosecond oscillator with abeam delivery system, which has an appropriate numerical aperture (NA)to confine density of the laser generated free electrons below thecritical value needed for formation of dense plasma, as illustrated inFIGS. 18A and 18B.

FIG. 18A graphs the average lasing energy vs treatment lasing irradiancelevel curves. The optical breakdown is approximately 1.0×1013 W cm−2 andthe corresponding average energy intensity for the applied laser wouldbe 0.319 W while the treatment applied an average energy 1.1525 nJ andthe corresponding irradiance level is around 0.19×10¹³ W cm⁻².

FIG. 18B graphs the objective numerical aperture vs treatment lasingirradiance level curve. The optical breakdown is approximately 1.0×10¹³W cm⁻² and numerical aperture required to achieve optical breakdown attreatment irradiance is 1.384.

In such a scenario, low-density plasma is formed, and treatment isreduced to ionization and dissociation of the water content within thefocal volume. This treatment results in production of reactive oxygenspecies. Initially, ionization of the water molecule occurs, and theejected electron is quickly hydrated resulting in formation of asolvated electron. Further, the cation radical of water, H₂O+, is ratherunstable and react with a water molecule producing hydrogen ion H₃O⁺,and hydroxyl radical OH*. Concurrently dissociation of the excited watermolecule occurs H₂O*^(→)H+OH*.

These are the primary reactions which occur within ˜10⁻¹³ seconds, andare followed by secondary reactions in which H, O₂ ⁻, OH⁻, H₂, H₂O₂, HO₂and other species, including singlet oxygen, are formed. In this studywe have shown that similar effects are achieved when femtosecondoscillator is employed. Since the energy of a photon at 1059 nmwavelength is 1.17 eV, six photons are required to interactsimultaneously with a bound electron to overcome the band gap of 6.5 eV,and produce electron-hole pair. The two-photon ionization is substitutedwith MPI, however, the nature of the chemical reactions and the radicalsproduces are likely the same.

FIG. 19 depicts results obtained via electron paramagnetic resonance(EPR) spectroscopy. FIG. 19A shows the results for femtosecond lasertreated DMPO solution, while FIG. 19B illustrates a control sample. Dueto short halflife of reactive oxygen species, spin-trapping wasemployed. Spin-trap reagent 5,5-dimethy-1-pyrroline-N-oxide (DMPO)solved in Dulbecco's phosphate-buffered saline (DPBS) has trapped OH*and O₂ ⁻, created after the solution was ionized with femtosecondoscillator. The radicals of interest (hydroxyl radicals and superoxideanion radicals) are identified through an adduct, which is a product oftheir reaction with DMPO.

Measurements of Temperature Distribution and Refractive Index

Formation of radicals enables treatment of ocular tissue through theirreaction with amino acids present in collagen fibrils. However, laserenergy driven creation and subsequent acceleration of the free electronsalso increases their kinetic energy, which is transferred to thesurrounding particles via collisions and non-radiative recombination.The process results in the heating of the plasma within the focalvolume. The energy density deposited into the focal volume is thereforeproduct of the total number of free electrons produced by the pulse andthe mean energy gain of each electron. Latter can be described as thesum of ionization potential and average kinetic energy. It is assumedthat the focal volume has the shape of Gaussian ellipsoid, and thus, thespatial distribution of the energy density within the focal volumefollows Gaussian distribution.

Significant rise of temperature within the focal volume and itsimmediate vicinity could have adverse effects. The one effect would berelated to generation of a thermoelastic tensile stress wave, which is afunction of the temperature distribution within the focal volume as wellas the shape of the focal volume. Temperature rise due to the lasertreatment could enable formation of a compressive stress wave, whichpropagates though the surrounding tissue. Thermal expansion is followedby tensile stress wave, governed by inertial forces, which travels inthe opposite direction. If the amplitude of the tensile stress exceedsthe tensile strength of the target material, a transient cavitationbubble will be formed regardless the fact that the optical breakdown hasnot been reached. The cavitation bubble could disrupt the collagenfibril arrangement resulting in localized change of refractive index(‘corneal haze’).

Another effect would be denaturation of collagen at elevatedtemperatures. Collagen denaturation transition occurs at 58° C. and themain denaturation transition occurs at 65° C. FIG. 20 illustrates atemperature distribution as a function of distance from the focal volumeof an embodiment. Temperature measurements illustrated in FIG. 20 showthat the maximum temperature increase is about 8° C., which remainsconstant within about 120 μm radius from the focal volume, after whichit decreases sharply. As typical temperature of a human eye is around35° C., the corneal treatment is very unlikely to cause any collagendenaturation.

Corneas were also treated to investigate whether change in refractiveindex would occur as a consequence of disruption of collagen fibrils.Post-treatment images are illustrated in FIG. 21 and show no differencein the refractive index between treated region and the surroundingcorneal tissue. Boxed region in the center has been treated withfemtosecond laser following protocol above. Both stereographic andNormaski contrast micrographs show no difference in refractive indexbetween treated and surrounding tissue.

The method and apparatus for cross-linking in cornea is applicable toother tissues, such as cartilage. As demonstrated through experimentsdescribed below, cross-linking was induced in cartilage. Inducingcross-linking in cartilage through femtosecond laser irradiation can beused to treat osteoarthritis, especially early osteoarthritis.Osteoarthritis (OA) is a severe degenerative disease with limitedtreatment options. Adult cartilage is an avascular connective tissuewith an extracellular matrix (ECM) consisting of collagens (COL) andproteoglycans (PGs), with the former providing tensile strength and thelatter being responsible for the compressive stiffness. Disruption ofthe COL network compromises the ability of cartilage to resist theswelling pressure of PGs, resulting in increased water content,decreased compressive stiffness, and greater vulnerability toprogressive cartilage degeneration and loss of function. The onset of OAis characterized by changes in the structure of the collagen network,but not necessarily its content. Crosslinks stabilize the COL fibernetwork, and their disruption leads to loss of tensile strength andstructural integrity of the bulk tissue. Targeted cross-linking of COLmatrix is a pathway for cartilage repair and an impediment of OAprogression.

The disclosed methods, devices, and systems for treatment of OA realizedby the induction of crosslinks (CxLs) in the COL fiber network viaoptical interaction, for example, femtosecond laser irradiation. Asexplained above, the cross-linking can be induced by direct absorptionof the radiated energy by the cartilage, or by inducing ionization inthe aqueous medium in and around the cartilage. Newly induced CxLs maystabilize the COL network and therefore enhance the mechanicalproperties of OA-afflicted cartilage.

Experiments have successfully demonstrated that a femtosecond oscillatoroperating in low-density plasma regime is capable of enhancing themechanical properties of corneal tissue, which predominantly consists oftype I COL, as well as articular cartilage.

The following experiments were used to validate the treatment ofcartilage with femtosecond laser irradiation for inducing cross-linking.Cartilage explants 5 mm round by 1.6 mm and 3.0 mm thickness wereobtained using a 3D printed slicer. The cartilage pieces were harvestedfrom three immature bovine proximal tibias with their articular surfacesintact. The treatment was performed with a Nd:Glass High-Q Femtosecondlaser oscillator system (temporal pulse width of 99 fs and 52.06 MHzrepetition rate) coupled with a 3-axis translational stage (Thorlabs,Inc.). The output wavelength was centered around 1060 nm, and the highnumerical aperture objective (Zeiss, Plan Neofluar 40×/0.6) deliveredabout 60 mW at the focal point. The treatment consisted of applyinglaser pulses by moving the stage in a x-y plane such that the laser pathfollowed a zigzag pattern at a feed rate of 2.2 mm/s, thus treating aplanar surface at the specific depth. The treatment was repeated atdifferent depths, effectively inducing ‘treatment layers’. However, inembodiments the laser treatment can be applied through an optical guideto deliver the light to a patient. In an exemplary embodiment, the lasertreatment is provided through an endoscope.

In the experiments, multiple treatment layers parallel to thesuperficial surface were applied with 50 μm distance between twoconsecutive planes. The specimens were gently inserted in a custom madeholder with 5 mm holes and kept moisturized during the treatment in aPBS bath. Two batches of experiments were carried out in this study,with each batch executed on a different joint. In the first batch, sixspecimens were treated with the femtosecond laser, each requiring 1hour, and was paired with an untreated control specimen that was placednext to the treated sample in the identical holder. All conditionsexcept the laser treatment were the same for the paired controls andtreated samples during the test. Two additional samples were used asfresh controls. In the second batch, five controls and five treatedsamples were used. Three of the treated samples received five lasertreated layers. The remaining two specimens were exposed to ten lasertreated layers. Cartilage explants were tested in a custom device underunconfined compression, using a creep tare load (0.1 N, 400 s) followedby stress-relaxation to 10% strain (0.5 μm/s ramp, 1800 s). Theequilibrium Young's modulus (Ey) was calculated from the explantcross-sectional area, the equilibrium load and the displacement. One-wayANOVA analysis was performed to analyze data.

The results of the experiments confirm the formation of cross-links inthe cartilage. In the first batch of experiments, laser treated sampleswere stiffened about 21% in comparison to the controls (p<0.003). Bothpaired and fresh controls had similar Young's modulus (FIG. 1 a ). Thesecond batch of experiments has shown similar stiffening of thespecimens treated with five layers in comparison to the controls(p<0.05). However, the samples treated with ten layers showed asignificant decrease in Ey (p<0.001, FIG. 1 b ).

Collagen (COL) is the major structural protein of most connectivetissues. The structural integrity and mechanical properties of articularcartilage are significantly affected by collagen cross-links, chemicalcompounds that connect COL fibrils as well as molecules within thecollagen. When a femtosecond oscillator operates in a regime below theoptical breakdown threshold, a low density plasma is created within thefocal volume. This plasma is not sufficiently energetic to produce ashock wave, and thus the interaction between the laser and the affectedtissue is photochemical, which leads to ionization of the matter withinthe focal volume and in its vicinity. Radicals produced by theionization field interact with the COL fibrils, which in turn produceCxLs. Therefore, laser induced CxLs are responsible for stiffening ofthe cartilage, which in turn yields enhancement of mechanical propertiesillustrated in FIGS. 22A and 22B.

Free radicals (or reactive oxygen species) created by the multiphotonionization are responsible for COL CxLs within articular cartilage. Theexperiments confirm that ultrafast irradiation with infrared (IR) laserpulses ionize water molecules in the target tissue. The recognition thatcross-linking can be achieved by ionizing water molecules, rather thanionizing the target tissue directly, enables the use of lasers atwavelengths that do not directly form covalent bonds in the collagen.Thus, wavelengths are selected to ionize water molecules and generatereactive oxygen species. The reactive oxygen species in turn inducecross-linking in the collagen of the treated cartilage. In anembodiment, the interaction mechanism is multiphoton, rather thantwo-photon ionization. This allows treatment with IR, and otherwavelengths, rather than ultraviolet (UV) pulses and much lower pulseenergies. Ionization of the water content within the focal volume in theinterior of the articular cartilage yields hydroxyl radicals, OH* andhydrogen ions H₃O+. Singlet oxygen may also be produced among otherspecies. Free radicals interact with the COL matrix producingcross-links.

These newly formed cross-links can be different from the ones thatnaturally occur in ECM, such as hydroxylysylpyridinoline. For example,one of the CxLs formed can be 1,3-dityrosine.

The disclosed study introduces a novel treatment of early OA anddetermines effectiveness of femtosecond laser treatment in delaying theprogression of collagen fatigue failure using a well-characterized invitro damage model of mechanically induced OA in both vital anddevitalized articular cartilage.

FIG. 22A shows results of mechanical property characterization of lasertreated samples, paired controls and fresh controls. FIG. 22B showsresults of mechanical property test of controls, five layers treatedsamples and ten layers treated samples. (*p<0.05: statistical changefrom corresponding initial value.)

In an exemplary embodiment of the disclosed subject matter, a method ofinducing cross-linking in a tissue containing water includes generatingreactive oxygen species by ionizing water molecules, the ionizingincluding focusing light on a tissue containing water. In the exemplaryembodiment, the focusing and intensity of the light is sufficient tocause ionization of water without causing optical breakdown of moleculesof the tissue. In the exemplary embodiment, the range of frequencies ofthe light is selected to excite water molecules without directly formingcovalent bonds.

In an exemplary embodiment of the disclosed subject matter, a method ofinducing cross-linking in a tissue containing water includes generatingreactive oxygen species from water molecules, the generating includingfocusing infrared light on a tissue containing water. In the exemplaryembodiment, the focusing and intensity of the infrared light issufficient to cause ionization of water without causing opticalbreakdown of molecules of the tissue and the range of frequencies of theinfrared light are selected to excite water molecules such thatcross-linking of collagen is caused by the reactive oxygen speciesrather than by the formation of covalent bonds.

In an exemplary embodiment of the disclosed subject matter, a method ofinducing cross-linking in a tissue containing water includes formingcross-links locally in collagen in the tissue by means of reactiveoxygen species by generating reactive oxygen species from watermolecules, the generating including focusing infrared light on a tissuecontaining water at an intensity and range of frequencies effective toionize water without the formation of covalent bonds and withoutinducing optical breakdown.

In an exemplary embodiment of the disclosed subject matter, a system forreshaping curvature of a region of a cornea having an initial curvatureincludes illumination optics configured to project an illuminationpattern onto at least a portion of the cornea. In the exemplaryembodiment, a camera is configured to record a pattern reflection fromthe at least a portion of the cornea and a control system, coupled tothe camera, is configured to convert the pattern reflection to a cornealtopography, and to compare the corneal topography to a desired cornealtopography to determine a deformation map. In the exemplary embodiment,a laser system is configured to induce ionization in the region of thecornea according to the deformation map to reshape the region from theinitial curvature to a new curvature. In the exemplary embodiment, acoupling device may be configured to stabilize the laser system withrespect to the cornea.

In an exemplary embodiment of the disclosed subject matter, an apparatusfor adapting a laser system for reshaping curvature of a region of acornea having an initial curvature includes a control system, adapted tobe coupled to the laser system and configured to compare an existingcorneal topography of at least a portion of the cornea to a desiredcorneal topography to determine a deformation map. In the exemplaryembodiment, laser modification optics are coupled to the control systemand configured to adjust laser output of the laser system, to modify aregion of the cornea according to the deformation map.

In an exemplary embodiment of the disclosed subject matter, a method ofreshaping curvature of a region of a cornea having an initial curvatureincludes inducing partial ionization in a region of the cornea byapplying laser light energy below optical breakdown.

In an exemplary embodiment of the disclosed subject matter, a method ofinducing cross-linking in tissue includes inducing ionization in aregion of the tissue by applying laser light energy below the opticalbreakdown level at wavelengths effective to generated reactive oxygenspecies in water without forming covalent bonds in collagen.

In an exemplary embodiment of the disclosed subject matter, a system forreshaping curvature of a region of a cornea having an initial curvatureincludes illumination optics configured to project an illuminationpattern onto at least a portion of the cornea, a camera configured torecord a pattern reflection from the at least a portion of the cornea,and a control system, coupled to the camera, configured to convert thepattern reflection to a corneal topography, and to compare the cornealtopography to a desired corneal topography to determine a deformationmap. In the exemplary embodiment, a laser system is configured to induceionization in the region of the cornea according to the deformation mapto reshape the region from the initial curvature to a new curvature, acoupling device is configured to stabilize the laser system with respectto the cornea, and the laser system generates a range of frequencies oflight selected to excite water molecules such that cross-linking ofcollagen is caused by the reactive oxygen species generated therebywithout directly forming covalent bonds. In the exemplary embodiment,the laser system has focusing optics that generate a maximum intensitythat is lower than a level that would produce optical breakdown in thehuman cornea.

In an exemplary embodiment of the disclosed subject matter, an apparatusfor adapting a laser system for reshaping curvature of a region of acornea having an initial curvature includes a control system, adapted tobe coupled to the laser system and configured to compare an existingcorneal topography of at least a portion of the cornea to a desiredcorneal topography to determine a deformation map. In the exemplaryembodiment, laser modification optics are coupled to the control systemand configured to adjust laser output of the laser system, to modify aregion of the cornea according to the deformation map. In the exemplaryembodiment, the laser modification optics generate light of a predefinedfrequency range and include focusing optics sufficient to produceintensity of laser light below the optical breakdown level sufficient toionize water without generating covalent bonds in collagen.

In an exemplary embodiment of the disclosed subject matter, a method ofchanging the mechanical properties of tissues containing collagen,includes focusing light on living tissue to generate reactive oxygenspecies from water in a tissue without directly generating covalentbonds in collagen in the tissue.

In an exemplary embodiment of the disclosed subject matter, a method oftreating tissue includes irradiating aqueous media in or surrounding thetissue with a laser at an energy level that avoids breakdown in thetissue until reactive oxygen species are produced, and inducingcross-linking in tissue with the produced reactive oxygen media.

In an exemplary embodiment of the disclosed subject matter, a system fortreating cartilage, the system includes a laser system, configured toinduce ionization in a region of the cartilage according to a treatmentpattern, the laser system generating a range of frequencies of lightselected to excite water molecules such that cross-linking of collagenis caused by the reactive oxygen species generated thereby withoutdirectly forming covalent bonds, the laser system having focusing opticsthat generate a maximum intensity that is lower than a level that wouldproduce optical breakdown in the human cornea.

In an exemplary embodiment of the disclosed subject matter, a method ofreshaping a patient's cornea from a first shape to a second shape,includes irradiating the cornea with a laser light in the absence of aphotosensitizer in or on the cornea, the laser light having energysufficient to cause ionization of water without causing opticalbreakdown of molecules of the tissue. In the exemplary embodiment, themethod also includes generating reactive oxygen species by ionizingwater molecules in or on the cornea, and inducing cross-linking in thecornea by the generated reactive oxygen species, wherein the inducedcross-linking changes the shape of the cornea from the first shape tothe second shape.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the measuring of the topography further includesprojecting an illumination pattern on the cornea, recording a patternreflection from the cornea with a camera, and converting the patternreflection into the topography of the cornea.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the tissue is a cornea, and the focusing includesprojecting an illumination pattern on the cornea.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the focusing includes scanning a laser over a region ofa cornea to be modified.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the scanning includes scanning a femtosecond laser.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the scanning includes scanning a femtosecond laserhaving an average power output from about 10 mW to about 100 mW.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the scanning includes scanning a femtosecond laserhaving a pulse energy of from about 0.1 nJ to about 10 nJ.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the scanning includes scanning in a pattern of exposurecomprising a circle, annulus, and/or ellipse.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the scanning includes scanning in multiple layers of thecornea.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the focusing includes projecting an illumination patternon the cornea.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter the focusing includes scanning a laser over a region of acornea to be modified.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter the scanning includes scanning a femtosecond laser.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the laser system is configured to cross-link collagen inthe cornea according to the deformation map.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the laser system comprises a femtosecond laser.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the femtosecond laser comprises a Nd:Glass femtosecondlaser.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the femtosecond laser comprises a laser having a pulsewidth of from about 50 fs to 150 fs.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the femtosecond laser comprises a laser having anaverage power from about 10 mW to about 100 mW.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the femtosecond laser comprises a laser adapted to emitlight in the wavelength range from about 600 nm to about 1600 nm.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the femtosecond laser comprises a laser adapted to emitlight in the infrared frequency range.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the laser system comprises a high magnificationobjective lens and a galvanometer configured to raster a laser beam.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the laser system further comprises an attenuator.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the laser modification optics are configured tocross-link collagen in the region of the cornea according to thedeformation map.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the laser modification optics further comprise anattenuator to reduce laser output power.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the illumination pattern comprises a pattern generatedby a continuous wave laser.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the new curvature corresponds with the desiredtopography.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the region of the cornea to be modified is based atleast in part on a deformation map.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the inducing partial ionization comprises scanning alaser over the region of the cornea to be modified.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the scanning comprises scanning a femtosecond laserhaving an average power output from about 10 mW to about 100 mW.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the femtosecond laser has a pulse energy of from about0.1 nJ to about 10 nJ.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the majority of the laser power is in wavelengths, orintegral fractions thereof, that are not absorbed directly by aminoacids in the collagen.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the majority of the laser power is in wavelengths, orintegral fractions thereof, that are absorbed directly by water to formreactive oxygen species.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the tissue is cartilage, and the applying the laserlight includes projecting an illumination pattern on the cartilage.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the inducing ionization comprises scanning a laser overa region of cartilage to be modified and the inducing is effective togenerate reactive oxygen species as a result of multiphoton interactionwith water.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the laser light has a wavelength in the infrared regionof the spectrum.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the laser has a range of wavelengths with most of thepower at wavelengths that are not directly absorbed by collagen orintegral multiples thereof.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the scanning comprises scanning a pulsed laser.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the laser is irradiated on the tissue in the absence ofa photosensitizer.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the femtosecond laser includes a laser adapted to emitlight in the wavelength range from about 600 nm to about 1100 nm.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the laser system includes a high magnification objectivelens and a galvanometer configured to raster a laser beam.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, measuring of the topography further includes projectingan illumination pattern on the cornea, recording a pattern reflectionfrom the cornea with a camera, and converting the pattern reflectioninto the topography of the cornea.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the illumination pattern includes a pattern generated bya continuous wave laser.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the new curvature corresponds with the desiredtopography.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the tissue being treated by the laser lacks aphotosensitizer.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the irradiating includes inducing low-density plasma inthe tissue and thereby ionizing one or more water molecules in theaqueous media such that at least an electron is ejected from the one ormore ionized water molecules.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the irradiating includes outputting pulses of the laserwith a duration of each pulse shorter than 1000 femtoseconds.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the system includes an optical pathway for guiding thelaser light emitted from the femtosecond laser to the cartilage.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the optical pathway includes an endoscope.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the second shape has a steeper curvature than the firstshape.

According to any of the foregoing exemplary embodiments of the disclosedsubject matter, the second shape has a less steep curvature than thefirst shape.

It has, thus, been shown that femtosecond oscillator can ionize targetmaterial under loose conditions, which enables applications intransparent dielectrics on a micro-scale. When operating below opticalbreakdown threshold, femtosecond laser produces low-density plasma, andthus generates reactive oxygen species (also referred to as freeradicals herein) within the focal volume. Newly formed radicals quicklyreact with the surrounding media and alter its chemical composition.When the surrounding media includes collagen fibers, collagencross-linking occurs. Such treatment regime is suitable for treatmentorganic transparent dielectrics such as corneal tissue. Radicals reactwith amino acids in the collagen triple helix to form crosslinks, whichenhances mechanical properties of corneal stroma.

The description herein merely illustrates the principles of thedisclosed subject matter. Various modification and alterations to thedescribed embodiments will be apparent to those skilled in the art inview of the teachings herein. Accordingly, the disclosure herein isintended to be illustrative, but not limiting, of the scope of thedisclosed subject matter.

The invention claimed is:
 1. A method of inducing cross-linking in a cartilage, the method comprising: generating reactive oxygen species from water molecules present in the cartilage by ionizing the water molecules within the cartilage, without a photosensitizer present in or on the cartilage, with a focused light at infrared frequencies irradiated on the cartilage, wherein the focused light at infrared frequencies is emitted by a femtosecond laser having an average power output from 10 mW to 100 mW, a pulse energy from 0.1 nJ to 10 nJ and a pulse width from 50 to 150 femtoseconds, focusing and intensity of the focused light at the infrared frequencies causes ionization of water without causing optical breakdown of molecules of the cartilage, and the focused light at the infrared frequencies includes a range of frequencies that excite the water molecules without directly forming covalent bonds in collagen.
 2. The method of claim 1, wherein the focused light at the infrared frequencies projects an illumination pattern on the cartilage.
 3. The method of claim 1, wherein the focused light at the infrared frequencies scans the femtosecond laser over a region of the cartilage to be treated.
 4. The method according to claim 1, further comprising: splitting a beam of the femtosecond laser to multiple scanning objectives, wherein the focused light is emitted by a laser head with the multiple scanning objectives.
 5. A method of inducing cross-linking in cartilage, comprising: inducing ionization in a region of the cartilage by applying laser light energy below an optical breakdown level at wavelengths effective to generated reactive oxygen species in water contained within the cartilage without directly forming covalent bonds in collagen.
 6. The method of claim 5, wherein: the inducing ionization comprises scanning a laser over a region of cartilage to be treated; and the inducing is effective to generate reactive oxygen species as a result of multiphoton interaction with water.
 7. The method of claim 6, wherein the laser light has a wavelength in an infrared region of the spectrum.
 8. The method of claim 6, wherein the laser has a range of wavelengths with most of its power at wavelengths that are not directly absorbed by collagen or integral multiples thereof.
 9. The method of claim 6, wherein the scanning comprises scanning a femtosecond laser having an average power output from about 10 to about 100 mW.
 10. The method of claim 6, wherein the scanning comprises scanning a femtosecond laser having a pulse energy of from about 0.1 nJ to about 10 nJ.
 11. A system for treating cartilage, the system comprising: a laser system, configured to induce ionization in a region of the cartilage according to a treatment pattern, the laser system generating a range of frequencies of light selected to excite water molecules such that cross-linking of collagen is caused by reactive oxygen species generated by the light without directly forming covalent bonds in collagen contained in the cartilage, the laser system having focusing optics that generate a maximum intensity that is lower than a level that would produce optical breakdown in the cartilage.
 12. The system of claim 11, wherein the laser system comprises a femtosecond laser.
 13. The system of claim 12, wherein the femtosecond laser comprises a Nd:Glass femtosecond laser.
 14. The system of claim 13, wherein the femtosecond laser comprises a laser having a pulse width of from about 50 to 150 fs.
 15. The system of claim 13, wherein the femtosecond laser comprises a laser having an average power from about 11 to about 100 mW.
 16. The system of claim 13, wherein the femtosecond laser comprises a laser adapted to emit light in a wavelength range from about 600 nm to about 1100 nm.
 17. The system of claim 13, wherein the femtosecond laser comprises a laser adapted to emit light in an infrared frequency range.
 18. The system of claim 13, further comprising an optical pathway for guiding the laser light emitted from the femtosecond laser to the cartilage.
 19. The system of claim 18, wherein the optical pathway includes an endoscope.
 20. The system of claim 11, wherein the laser system further comprises an attenuator. 